Double-Stranded RNAs High-Efficiently Protect Transgenic Potato

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Double-Stranded RNAs High-Efficiently Protect Transgenic Potato from Leptinotarsa decemlineata by Disrupting Juvenile Hormone Biosynthesis

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 11/07/18. For personal use only.

Wenchao Guo,†,‡,# Chao Bai,†,⊥,# Zhian Wang,†,§,# Peng Wang,†,§ Qiang Fan,†,∥ Xiaoxiao Mi,∥ Le Wang,† Jiang He,‡ Jinhuan Pang,† Xiaoli Luo,§ Weidong Fu,†,⊥ Yingchuan Tian,† Huaijun Si,∥ Guoliang Zhang,*,†,⊥ and Jiahe Wu*,† †

State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Institute of Plant Protection, Xinjiang Agricultural Academy of Sciences, Xinjiang, Urumqi, China § Institute of Cotton Research, Shanxi Agricultural Academy of Sciences, Shanxi, Yuncheng, China ∥ College of Biology Science and Technology, Gansu Agricultural University, Gansu, Lanzhou, China ⊥ Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China ‡

S Supporting Information *

ABSTRACT: RNA interference (RNAi) has been developed for plant pest control. In this study, hairpin-type double-stranded RNA (dsRNA) targeting the juvenile hormone (JH) acid methyltransferase (JHAMT) gene (dsJHAMT) was introduced in potato plants via Agrobacterium-mediated transformation. The results indicated that the transcriptional RNA of dsJHAMT accumulated in the transgenic plants. The transcripts and proteins of the L. decemlineata JHAMT gene were significantly reduced in larvae feeding on dsJHAMT transgenic foliage. The dsJHAMT had a significant negative effect on the growth and development of L. decemlineata, especially resulting in less oviposition. Importantly, in the field trials, transgenic plants are highefficiently protected from insect damage mainly because surviving insects laid fewer or no eggs. Even full protection from beetle damage can be acquired by continuously lowering insect population size at large scale in the field over the years. Therefore, the transgenic plants expressing dsJHAMT successfully provided an additional option for plant pest control. KEYWORDS: double-stranded RNAs, juvenile hormone acid methyltransferase (JHAMT), Leptinotarsa decemlineata Say., pest control, RNA interference, transgenic potato

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INTRODUCTION The Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), a notorious agricultural pest, defoliates the solanaceous family plants, such as potato (Solanum tuberosum), tomato (Solanum lycopersicum), Solanum nigrum, and so on. The potato and tomato, two important crops, are severely damaged by this insect pest, leading to a large yield loss worldwide. The protection of potato production from this pest has mainly relied on insecticides. However, all major insecticide classes have failed to control CPB damage because of the development of resistance.1 Additionally, the indiscriminate use of insecticides caused the resurgence of nontarget pests, destruction of beneficial insects, and food and environmental contamination.1 Therefore, exploring alternative strategies to control the damage of CPB is desirable to protect solanaceous family plant production worldwide. RNA interference (RNAi) is an effective gene-silencing mechanism in eukaryotes,2 and the introduction of homologous double-stranded RNA (dsRNA) to insect has been developed as an effective insect resistant system in plants.3,4 Environmental RNAi (eRNAi) or plant-derived dsRNA fed to insects is absorbed through midgut cells, which is then processed into small interfering RNAs (siRNAs) by the endoribonuclease Dicer.5−7 Thus, dsRNAs become highly species-specific insecticides by targeting specific insect genes.8 © XXXX American Chemical Society

Recently, many studies confirmed that transgenic plant-derived dsRNA significantly decrease targeted gene mRNA levels, resulting in larval developmental deformity and lethality and efficiently controlling pest damage to plants.9−17 These data strongly suggest that transgenic plant-derived dsRNA is emerging as a powerful approach to protect plants from damage by insect pests. However, although these transgenic plants expressing dsRNAs targeted against insect genes are resistant to pests because they impair insect growth and development, high-efficient or even full protection of plants from insect pests in the field has not yet been achieved. Therefore, additional studies of transgenic plant-derived dsRNA targeted against insect genes are necessary. Juvenile hormone (JH), a sesquiterpenoid present in insects, regulates CPB growth and development during its life cycle.18,19 JH analogs, including pyriproxifen (Pyr), methoprene, and hydroprene, were synthesized and shown to effectively control the insect molting process. 19 The application of hormone analogs is a new strategy for integrated pest management.20 Essential genes involved in hormone Received: July 27, 2018 Revised: October 18, 2018 Accepted: October 26, 2018

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DOI: 10.1021/acs.jafc.8b03914 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

by electroporation. The genetic transformation of potato was performed as previously reported.29 PCR Detection for Putative Transgenic Potato Plants. The young leaves of the transgenic potato and wild-type plants were selected for genomic DNA extraction for PCR detection and Southern blot analysis. Total DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to manufacturer’s protocol. The specific primers for PCR detection in dsJHAMT transgenic plants are shown in Table S1. Southern Blot Analysis for Transgenic Potato Plants. To investigate the copy number of dsJHAMT insertion in transgenic potato plants, Southern blot analysis was performed according to the method of Ahmad et al.30 Briefly, after a total of 10 μg of DNA was digested with Hind III, it was separated by agarose gel electrophoresis. The DNA in the gel was transferred to an Amersham Hybond-N+ positively charged nylon membrane (GE Healthcare UK Limited, Little Chalfont, UK) by using capillary transfer overnight. The PCR product of the JHAMT fragment probe was labeled with [α-32P] dCTP using a random primer DNA labeling kit (Promega, Madison, WI). RT-PCR and qPCR Analyses for Transgenic Potato Plants. Total RNA was extracted from young leaves of transgenic potato and control plants using a plant RNA extraction kit (Invitrogen, Carlsbad, CA), and then any contaminating genomic material was removed with RNase-free DNase I (Invitrogen). A total of 2 μg of RNA was used for reverse transcription according to the manufacturer’s instructions (Invitrogen) using random hexamer primers (Table S1). The dsJHAMT transcript levels of the transgenic plants were monitored using semi-RT-PCR and qPCR techniques. For semi-RT-PCR analysis, 0.5 μL of first-strand cDNA and a pair of specific primers (Table S1) were used. The products were performed by the normal PCR protocol. Actin gene was amplified by 20 cycles for internal reference correction, and target genes were done by 30 cycles. All qPCR tests were performed with an ABI Prism 7500 real-time PCR system using a 25 μL mixture containing 10 ng of synthesized cDNA, 1× iQ SYBR green supermix, and 0.2 μM forward and reverse primers for the target genes (Applied Biosystems, Foster City, CA, USA). The potato actin1 gene was used as an internal control. To calculate relative expression levels, serial dilutions (0.2−125 ng) were used to produce standard curves for each gene. PCRs were carried out in triplicate using 96-well optical reaction plates, comprising a heating step for 3 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 58.5 °C for 1 min, and 72 °C for 20 s. Amplification specificity was confirmed by melt curve analysis on the final PCR products in the temperature range 50−90 °C with fluorescence acquired after each 0.5 °C increment. The fluorescence threshold value and gene expression data were calculated using the CFX96 system software. Values represent the mean of three real-time PCR replicates ± SD. Northern Blot Analysis for Transgenic Potato Lines. Total RNA from transgenic lines and the control were extracted as described above and used for Northern blot analysis. To investigate the RNA accumulation of dsJHAMT, 50 μg of total RNA was separated on 1.5% agarose gels containing 6% formaldehyde and transferred to nylon N+ membranes. For siRNA analysis, 30 μg of total cellular RNA was separated by electrophoresis on 15% PAGE gels and electrically transferred to nylon N+ membranes. The PCR product generated by amplification with specific dsJHAMT primers, which amplified the first 290 bp length of dsJHAMT fragments, shown in Table S1, was labeled with [α-32P] dCTP using a random primer DNA labeling kit (Promega). Bioassay. The foliage of dsJHAMT transgenic plants and the control were detached for the larvae feeding bioassay, which was continuously performed in 2014 and 2015. The newly ecdysed second- and fourth-instar larvae (Data S1) were used to evaluate the effects of dsJHAMT transgenic plants. To evaluate the rescue effects, 0.05 μg of the JH analogue pyriproxyfen, 2-[1-methyl-2-(4phenoxyphenoxy)ethoxy] pyridine (Ivy Fine Chemicals Corporation, Cherry Hill, NJ), was coated onto transgenic foliage for a larvae feeding assay.

biosynthesis are used as ideal targets for transgenic plantderived dsRNA to control pests.16 JHs are biosynthesized de novo in insects. First, farnesyl diphosphate is biosynthesized from acetyl-CoA through the classical mevalonate pathway. JH biosynthesis beginning with farnesyl diphosphate is unique to insects and crustaceans, and it is a successive catalyzed reaction using enzymes, including farnesyl diphosphate pyrophosphatase, farnesol oxidase, farnesal dehydrogenase, epoxidase, and a JH acid methyltransferase (JHAMT).21−23 JHAMT is the last rate-limiting enzyme of JH biosynthesis. Three reports showed that JHAMT overexpression in Drosophila melanogaster significantly prolongs pupal development.24 Conversely, knockdown of JHAMT reduced JH titer, resulting in larval death in Aedes aegypti.25 In CPB, JHAMT knockdown significantly affected insect growth.26 Therefore, JHAMT should be an ideal candidate gene to produce plant-derived dsRNA to protect plants from insect damage. In the present study, we isolated a JHAMT gene from L. decemlineata and constructed a plant expression vector by inserting a JHAMT fragment and its reverse complement into binary expression vector. The dsJHAMT transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. After the CPB larvae were fed transgenic potato foliage, both LdJHAMT transcripts and protein accumulation were significantly reduced, negatively affecting insect growth and development. Importantly, the surviving females lay fewer or no eggs, resulting in decreasing reproduction both in the laboratory and fields. Our results indicate that JHAMT may serve as a potential target for transgenic plant-derived dsRNA to high-efficiently protect plants from damage caused by CPB via the disruption of insect reproduction potential. Under continuous planting of these transgenic potatoes at large scale over the years, full protection from beetle damage can be developed in the field.

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MATERIALS AND METHODS

Plant Materials. The potato cv. “Favorita”, commercially available in China, was provided by Professor Peng Xuexian, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Its stem sections used as explants were prepared for Agrobacterium-mediated transformation. The transgenic plants were grown in a greenhouse at 25 ± 2 °C under a 14 h light/10 h dark cycle. Tubers were harvested for propagating next generation. The continuously twice-propagated transgenic plants were used for subjecting to experiments. The two CPB populations originated from Changji and Tekesi counties, which were reared on potato plants in a greenhouse at 25 ± 2 °C under a 14:10 h light/dark photoperiod for insect-resistant assays in the Institute of Plant Protection, Academy of Agricultural Sciences of Xinjiang Province. Gene Cloning, Vector Construction, and Potato Transformation. The target fragments in the coding sequence of the LdJHAMT gene (GenBank accession no. KP274881) were selected for RNAi targets as shown in Figure S1a. To construct the hairpin dsRNA vector, this fragment was amplified using specific primers (Table S1). The PCR products were digested by Xho I and EcoR I first and then were cloned into the pHANNIBAL vector, resulting in pHANNIBAL-sense vector. Pyruvate dehydrogenase kinase gene (PDK, 767 bp) intron in the sense orientation was introduced into pHANNIBAL-sense to obtain pHANNIBAL-sense-PDK. Subsequently this construct ligated with antisense digested by Hind III and BamH I to form pHANNIBAL-sense-PDK-antisense structure.27 Then, the Not I-digested expression cassette with hairpin structure fragment under control of the CaMV35S promoter was subcloned into the binary expression vector pART27.28 The resulting vectors were introduced into the Agrobacterium tumefaciens strain LBA4404 B

DOI: 10.1021/acs.jafc.8b03914 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Each Petri dish (9.0 cm in diameter) was maintained under laboratory conditions for insect rearing. The petioles of the detached leaves were enwrapped with soaked cotton and placed it into Petri dishes. The five second- or fourth-instar larvae were inoculated on the leaves until pupation, respectively. The potato foliage was replaced with corresponding fresh foliage each day. Each treatment was replicated 20 times (20 dishes). We kept them until they grow to be second- and fourth-instar larvae. The insects from six dishes were used to extract total RNA and protein, and the others were continuously fed the corresponding foliage. Dead larvae were found and recorded each day. The surviving larvae weight was individually assessed, and the pupation rates were recorded during a trial period according to standard criteria previously described.26 The adults were weighed, and the female was paired with two sexually mature males and inoculated onto a potato plant. Eggs laid by each female were counted and evaluated during the first 10 days after emergence. Corrected mortality was used for statistical analysis using the formula corrected mortality = [(mortality rate of the treatment group − mortality rate of the control group)/(1 − mortality rate of the control group)] × 100%. Three biological replicates were performed each year. Mean ± SD were separated using Student’s t-test at P < 0.01. qPCR Analysis of LdJHAMT Transcription Level in Tested Larvae. To analyze the effects of plant-derived dsJHAMT on the development of larvae, total RNA was extracted from larvae 3 days after the feeding bioassay using the SV Total RNA Isolation System kit (Promega). The transcriptional levels of LdJHAMT and Krüppel homologue 1 (LdKr-h1) were examined by qPCR using specific primers (Table S1). The internal control gene was ribosomal protein 18 (RP18) according to the method described by Li.31 The experiment was repeated three times. The data were analyzed using the 2−ΔΔCt method.32 Immunoassay of LdJHAMT Protein Accumulation. To further analyze the effects of plant-derived dsJHAMT ingestion, the JHAMT protein concentration of CPB larvae was measured by immunoassay. Total protein was extracted from larvae 3 days after feeding on dsJHAMT transgenic foliage and the control using the I-PER Insect Cell Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA). The JHAMT protein (AKQ00043.1) concentration in the samples was measured by the Bradford method. Approximately 20 μg of soluble protein was loaded into each well for immunoassay analysis. Recombinant JHAMT protein introduced by Escherichia coli was used to produce antibody. Rabbit antiserum against JHAMT (1:3000 v/v) prepared in our laboratory and alkaline phosphatase-conjugated goat antirabbit immunoglobulin (Ig) G (Promega; 1:5,000 v/v) was used as primary and secondary antibodies, respectively. JH Titer Measurement. To analyze the effects of plant-derived dsJHAMT ingestion on JH biosynthesis, the JH titer of larvae fed transgenic foliage was determined. Hemolymph collected from larvae was used to extract JH according to previously reported methods.33 The JH titer (ng mL−1 hemolymph) was quantified using liquid chromatography−tandem mass spectrometry (LC−MS) according to previously reported methods.33 Evaluation of the Resistance of dsJHAMT Transgenic Plants to CPB in the Field Trials. To evaluate the insect resistance of dsJHAMT transgenic plants in the field, artificial infestation with the second-instar CPB larvae was performed according to the method previously described by Zhou et al.29 Briefly, at a farm located in Changji county, Xinjiang Uygur Autonomous Region, China, in 2014 and 2015, dsJHAMT transgenic lines and control were inoculated with five s-instar CPB larvae originated from Changji insect population and shielded with a 0.833 mm aperture screen net to avoid insect transfer. In Tekesi county, the similar field trials were performed using the Tekesi CPB population in 2017. After 50 days, the damage symptoms of plants were observed, and the numbers of CPBs (i.e., survivors and offspring) and eggs were assessed in the field. The experiment was designed with three replicates. Evaluation of Agronomic Performance of Transgenic Potatoes in the Field. The three transgenic potato lines of dsJHAMT, L4, L5, and L6, and the nontransgenic control were evaluated for agronomic performance. This experiment was

performed on a farm of Tekesi country in Xinjiang Uygur Autonomous Region, in 2014 and 2015, where the potato was planted continuously for 3 years, resulting in moderate damage by CPB. Tuber seeds of each line were sown in a plot, which were grown in five rows of approximately 15 m2 in area, including approximately 120 plants. Each plot was shielded with a 0.833 mm aperture plastic net to avoid insect transfer among plots on the July first each year. Additionally, a supplementary nontransgenic control was grown, which was 2 m apart from the tested fields. The supplementary nontransgenic control underwent the same manipulation as the tested fields, except insecticide application. The experiment was performed in three replicates. The plant height, the branch number per plant, the tuber number per plant, the mean weight per tuber, and the yield per plot were measured at maturity. The data on the plant height, branches, tubers, tuber weight, and plot yield were analyzed by oneway ANOVA. The significant difference of years was first examined using the F-test. Mean values for each year were separated using Duncan’s multiple comparison test at P < 0.05.

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RESULTS Development and Molecular Analyses of dsJHAMT Transgenic Potato Plants. The full-length LdJHAMT cDNA was isolated according to the sequence deposited in GenBank (accession no. KP274881), and the target sequence from the first to the 290th nucleotide of this gene was selected according to the nucleotide identical analysis, which does not contain off-target sequences in NCBI data (Figure S1a). Then the 290 bp sense and antisense of target sequences were cloned into plant RNAi vector pHANNIBAL with PDK intron to form a dsRNA hairpin structure. This specific hairpin fragment was inserted into a binary expression vector pART27 under control of the constitutive CaMV35S promoter for plantderived dsJHAMT-mediated RNAi in insects (Figure S1b). Potato stem sections were used as explants for transformation, and the different shoots were induced and cultured into regenerated plantlets. A total of 85 putative transgenic potato plants were generated by using “Favorita” cultivar as explants mediated by Agrobacterium-mediated transformation methods, of which 62 independent positive transgenic plants were obtained according to the PCR analysis (Figure 1a). The PCRpositive plants were selected for Southern blot analysis. The results showed that approximately 50% of plants contained a single copy T-DNA insertion (Figure 1b). To examine the expression level of dsJHAMT in transgenic plants, RT-PCR and qPCR were performed. According to the results of the semi-RT-PCR analysis, transgenic plants expressed dsJHAMT, whereas the control did not. Furthermore, dsJHAMT expression levels were different between transgenic plants (Figure 1c). The results of the qPCR analysis confirmed that the expression level of dsJHAMT in transgenic plants was significantly different (Figure 1d). For example, the dsJHAMT expression level in plant L6 was 5.3-fold higher than in plant L1. To further monitor the dsJHAMT transcription in transgenic plants, Northern blot analyses were performed. The results showed that dsJHAMT were detected in the transgenic plants, although dsRNA can be readily processed into siRNA by an endogenous plant RNAi pathway (Figure 1e). Therefore, three transgenic plants with a relatively higher expression level of dsRNAs and comparable phenotypes to the wild-type plants, L4, L5, and L6, were selected for RNAi efficiency and insectresistant analysis (Figure 1f). Analysis of Endogenous LdJHMAT Transcription in Larvae Fed dsJHAMT Transgenic Foliage. To analyze the LdJHMAT RNAi in larvae ingesting plant-derived dsRNA, the C

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the control, which was associated with the LdJHAMT expression and protein accumulation in tested larvae (Figure 2c). To determine whether the JH signaling pathway in tested larvae was disrupted by plant-derived dsJHAMT ingestion, we examined the expression levels of Krüppel homologue 1 gene (Kr-h1), which is a JH early inducible gene in L. decemlineata and other insect species.24,25,29,34,35 Kr-h1 mRNA levels of the second- and fourth-instar larvae 3 days after feeding on dsJHAMT transgenic foliage were lowered by approximately 85 and 65% compared to the control, respectively (Figure 2d). When transgenic foliage was coated with the JH analog pyriproxifen, Kr-h1 expression levels were recovered compared to the control (Figure 2e). Together, these data indicate that low JH titers in JHAMT RNAi larvae resulted in reducing Krh1 mRNA levels and disrupting the JH signal pathway. Furthermore, application of pyriproxifen rescued Kr-h1 expression. Effects of Plant-Derived dsJHMAT Ingestion on CPB Growth and Development. To investigate the effects of plant-derived dsJHAMT ingestion on CPB, the growth and development of the second- and fourth-instar larvae fed dsJHAMT transgenic foliage were assessed. The results showed that the corrected mortalities of the second- and fourth-instar larvae continuously fed dsJHAMT transgenic foliage reach an average of 21.2% and 18.2%, respectively (Figure 3a). The corrected mortalities of CK were an average of 24.8% (Figure 3a). The surviving second- and fourth-instar larvae feedings on the transgenic foliage for 9 days grew significantly slower than the control and weighed an average of 36.4 and 52.1 mg per individual, respectively, whereas the control larvae fed wildtype foliage were 64.5 and 84.1 mg per individual (Figure 3b,c). Additionally, plant-derived dsJHAMT ingestion significantly affected larval duration and pupation. The larval duration of the second- and fourth-instar larvae fed transgenic foliage was 14.6 and 9.5 days, respectively, whereas the controls were 16.1 and 10.6 days, respectively (Figure 3d). Similarly, the pupation rates of the second- and fourth-instar larvae feedings on dsJHAMT transgenic foliage were 51.7 and 62.3%, respectively, whereas the control was 94.0 and 92.5%, respectively (Figure 3e). Additionally, to further explore the function of plant-derived dsJHAMT in tested larvae, exogenous pyriproxifen was used to coat the transgenic foliage to compensate for the JH deficit of the tested larvae. The results showed that pyriproxifen application rescued larvae from the negative effects of dsJHAMT transgenic foliage ingestion, resulting in the similar phenotypes as the control larvae fed wild-type foliage (Figure S3). The emergence rates of pupa from the second- and fourthinstar larvae fed dsJHAMT transgenic foliage was significantly reduced compared to the control, with only 45.8 and 52.6% of the control (Figure 4a), respectively. The average weights of the adults from the two treatments were 126 and 151 mg per individual, whereas the control weights were 193 and 205 mg per individual, respectively (Figure 4b). At the first 10 days post-emergence, two controls deposited on average 53 and 55 eggs per female, whereas the adult female from the second- and fourth-instar larvae fed dsJHAMT transgenic foliage lay fewer or no eggs (Figure 4c). Indeed, we found that only 2 of 33 female adults from the tested second-instar larvae laid a total of 46 eggs and that 3 of 52 females from the tested fourth-instar larvae laid 94 eggs. When exogenous pyriproxifen was used to coat the transgenic foliage, the results showed that this JH

Figure 1. Molecular analysis of dsJHAMT transgenic potato plants. (a) PCR analysis of dsJHAMT putative transgenic plants. Lane M, DNA marker; lane N, nontransformed plant; lane P, plasmid control; lanes 1−18, representative independent transgenic plants. (b) Southern blot analysis of transgenic potato and control plants. Lanes 1−10, representative transgenic plants. (c) RT-PCR analysis of dsJHAMT transgenic plants. Lanes 1−6, six transgenic plants; upper pattern shows dsJHAMT amplification bands, and lower pattern presents potato actin1 expression levels. (d) qPCR analysis of transgenic plants. L1 expression level is regarded as 1. (e) Northern blot analysis of dsJHAMT accumulation in transgenic potato plants. (f) Comparable phenotypes of the three candidate transgenic plants with a nontransformed plant.

second- and fourth-instar larvae fed transgenic and control plant foliage were evaluated. As shown in Figure S2, the damage to dsJHAMT transgenic and control foliage was similar after insect inoculation. The second- and fourth-instar larvae 3 days after feeding on dsJHAMT transgenic foliage had significantly reduced LdJHAMT expression, with only 3.5− 6.2% of the control expression level in the second-instar larvae and 24.3−34.5% of the control in the fourth-instar larvae (Figure 2a). Then, we examined LdJHAMT accumulation in the tested larvae with protein immunoblots. The results showed that LdJHAMT accumulation in the second- and fourth-instar larvae 3 days after feeding on transgenic foliage was significantly reduced compared to the control (Figure 2b). Taken together, LdJHMAT transcription levels and protein accumulation in larvae reared with transgenic foliage were significantly down-regulated by the transgenic plant-derived dsJHAMT. To further characterize the dsJHAMT in tested larvae, the JH titer was examined using LC−MS. JH titers of the second- and fourth-instar larvae 3 days after feeding on dsJHAMT transgenic foliage was significantly decreased compared to D

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Figure 2. Effects of plant-derived dsJHAMT ingestion on LdJHAMT expression, LdJHAMT accumulation, JH titer, and JH signaling pathway of CPBs. (a) The second- and fourth-instar CPB larvae fed on three transgenic and control foliage for 3 days (CK). (a) JHAMT transcriptional expression. (b) JHAMT protein accumulation. CBB: total protein of samples stained with Coomassie brilliant blue (CBB). (c) JH titer. (d, e) LdKr-h1 expression level (d) and that treated with pyriproxifen (Pyr) (e). The normalized values are the ratios of data from the larvae fed transgenic foliage to those of control larvae. The test was repeated three times. The error bars represent the SD of three biological replicates. Asterisks indicate statistically significant differences compared to the control, analyzed using Student’s t-test (P < 0.01).

Figure 3. Effects of plant-derived dsJHAMT ingestion on larval performance. The newly ecdysed second- and fourth-instar larvae fed three dsJHAMT transgenic lines or nontransformed line (CK). (a) The corrected mortalities of larvae are calculated from feeding on transgenic foliage to pupation. (b) Larvae weight at 9 day after treatment. (c) The phenotypes of larvae swept from the transgenic or control foliage. (d) Larval duration. (e) Pupation rates. Corrected mortalities to the control are calculated. The error bars represent the SD of three biological replicates. Asterisks indicate statistically significant differences compared to the control, analyzed using Student’s t-test (P < 0.05 or 0.01). Bar = 5 mm.

analog rescued larvae from the dsJHAMT RNAi effect (Figure S4). Together, these data reveal that insects ingesting plant-

derived dsJHAMT exhibit lower adult weights and pupa emergence rates compared to the control. Importantly, the E

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Figure 4. Effects of plant-derived dsJHAMT ingestion on adult performance. The second- and fourth-instar larvae fed three transgenic lines and nontransformed line (CK). (a) The emergence rates of adults. (b) The adult weights just after emergence. (c) Eggs per female during the first 10 days after emergence. The error bars represent the SD of three biological replicates. Asterisks indicate statistically significant differences compared to the control, analyzed using Student’s t-test (P < 0.01).

tested female adults laid fewer or no eggs, possibly resulting in a loss of reproduction potential. Resistance Analysis of dsJHAMT Transgenic Potato Lines against CPB in the Field. To evaluate resistance to CPB in two different locations, three dsJHAMT transgenic potato lines, L4, L5, and L6, were planted at a farm located in Changji county (2014 and 2015) and Tekesi county (2017) in Xinjiang Uygur Autonomous Region. Five second-instar larvae per plant from corresponding insect populations to Changji or Tekesi county were inoculated to both the transgenic and control lines, and each line was caged using a net to avoid pest transfer. In Changji County, damage symptoms of plants were observed, and the numbers of insects and eggs were counted 50 days after inoculation in 2014 and 2015. The results showed that in the control the foliage was consumed with a leafskeleton left only and that the average number of CPB per plant reached 138, whereas the three transgenic lines were only slightly damaged and the number of insects per plant was approximately 1.2, 2.2, and 1.8, respectively, which was significantly lower than the control (Figure 5a,b). Furthermore, we did not find any eggs in the three transgenic lines, whereas in the control plants an average 155.8 eggs per plant was observed. At 65 days after inoculation, the nontransgenic plants were completely destructed, whereas the transgenic plants showed high resistance to CPB (Figure 5c). In Tekesi county, the results of field experiments using corresponding CPB population in 2017 confirmed that the dsJHAMT transgenic plants showed higher resistance to CPB compared to the control (Figure S5). These results indicate that dsJHAMT transgenic plants are high-efficiently protected from CPB damage in the different fields, possibly because of the inhibition of CPBs reproduction. Evaluation of the Agronomic Traits of Transgenic Potato Lines. To evaluate the agronomic traits of transgenic potato lines, field trials were performed in 2014, 2015, and 2017. The results of three years of field trials were consistent, and the difference in the time of year was not significant (P > 0.05). The agronomic traits of the three transgenic lines and the control without insecticide application were significantly different each year (Table 1). The plant height, branches per plant, average tuber weight, and plot yield of the transgenic lines were significantly higher than those of the N control without insecticide spraying. Moreover, the agronomic performance of the three transgenic lines was similar to that of the supplementary nontransgenic control, which was sprayed with insecticides. Therefore, the three transgenic

Figure 5. Resistance of dsJHAMT transgenic lines to CPB in the field. (a) Damage symptoms of nontransformed and transgenic plants (L4 as a representation) by insects 50 days after inoculation. CK: nontransformed plants. (b) Average number of surviving CPBs 50 days after inoculation in the transgenic lines and control. The error bars represent the SD of three biological replicates. Asterisks indicate statistically significant differences compared to the control, analyzed using Student’s t-test (P < 0.01). (c) High resistance of dsJHAMT transgenic lines to CPB in the field. Photo was taken at 65 days after insect inoculation.

lines may be used as cultivars and germplasm for insectresistant breeding, subject to food and environmental safety assessments.

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DISCUSSION Transgenic Bt plants have been developed to control pest damage for two decades. To control CPB damage, the transgenic cry3A potato plants with a marker gene29 or marker free gene to control CPB damage have been produced.34 However, transgenic Bt plants have a specific insecticidal spectrum, and target insects may evolve resistance to the responding Bt gene.35,36 Therefore, a new strategy is necessary F

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Journal of Agricultural and Food Chemistry Table 1. Agronomic Traits of the Three Transgenic Potato Lines in the Fielda years/locations

lines

2014/Changji

L4 L5 L6 N Suppl. N L4 L5 L6 N Suppl. N L4 L5 L6 N Suppl. N

2015/Changji

2017/Tekesi

plant height (cm) 62.3 63.5 61.8 55.5 63.2 59.2 58.6 58.2 53.5 58.8 63.2 63.4 63.8 55.6 63.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.2 4.7 3.9 5.8 3.4 4.5 4.6 4.3 5.9 3.9 3.7 3.4 3.3 4.2 3.8

b b b a b b b b a b b b b a b

branches/plant 3.9 3.7 3.7 3.2 3.8 3.7 3.9 3.8 3.3 3.9 3.7 3.7 3.8 3.2 3.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.4 0.4 0.6 0.3 0.3 0.4 0.4 0.4 0.2 0.5 0.5 0.4 0.4 0.4

bc b b a b b bc b a bc b b b a b

tubers/plant 6.2 6.4 6.2 6.3 6.4 6.1 6.3 6.3 6.2 6.3 6.5 6.6 6.4 6.4 6.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.4 0.6 0.9 0.4 0.6 0.6 0.5 0.8 0.5 0.7 0.8 0.8 0.8 0.7

mean weight/tuber (g) 132.4 128.9 130.9 66.7 134.8 142.7 144.5 139.9 69.5 147.1 132.2 129.8 131.3 62.7 132.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

15.6 12.9 b 14.6 b 8.3 a 18.6 b 18.5 b 16.9 b 15.8 b 12.3 a 17.6 bc 16 b 15.3 b 13.9 b 10.6 a 13.1 b

yield/plot (kg) 92.6 95.3 93.1 48.9 97.5 105.2 108.9 104.7 52.1 110.5 112.4 110.3 111.6 53.3 113

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.2 b 7.9 b 7.7 b 6.4 a 5.4 b 7.6 b 8.2 b 6.8 b 4.4 a 4.9 b 13.6 b 13 b 11.8 b 9a 11.2 b

a

Note: A total of 20 plants for each plot (line) were evaluated, and the experiment was conducted in three replicates. The values are given as the mean ± SD. The values within each column followed by different letters are significantly different at P < 0.05. N: nontransgenic control without insecticide spraying. Suppl. N: supplementary nontransgenic control with insecticide spraying.

The phenotypes under JH deficiency in L. decemlineata were also observed for feeding on potato foliage coated with bacteria expressing dsJHAMT.26 RNAi-mediated knockdown of the JHAMT gene in two other reports indicated that the developing insects showed some malformation, such as precocious metamorphosis in T. castaneum and smaller basal oocytes in S. gregaria.22,46 Our results show that plant-derived dsJHAMT ingestion is biologically more effective in L. decemlineata than in T. castaneum and S. gregaria.22,46 These data also indicate that the sensitivity to RNAi may differ among insect species, which was previously reported.43−45,50−54 In the present study, transgenic potato plants with dsJHAMT can cause negative insect performance, including increased lethality, decreased weight, shortened developmental period, reduced pupation rate and emergence rate of pupae, and laid fewer or no eggs. Overall, the transgenic potato lines, L4, L5, and L6, only had partial success in L. decemlineata control in the laboratory; however, in the field trials located at two countries for three years, these transgenic plants containing dsJHAMT are high-efficiently protected from artificial infested insect damage possibly because surviving female adults lay fewer or no eggs and remarkably reduce reproduction potential. Indeed, 50 d after inoculation, the next generation insects (main offsprings of artificial inoculated second-instar larvae) laid lots of eggs in the control trials, an average of 155.8 eggs per plant (Figure 4). However, we hardly observed eggs on the transgenic plants when the sample plants were investigated at 50 day after CPB inoculation. Of course, some eggs may be missed during the survey, or eggs were not observed due to only a point-in-time searching (at 50 day after inoculation) and possibly found if the investigation was successively performed. Currently, transgenic dsRNA plants targeting a range of insect species4,9−15 are considered to partially protect against insect damage, excluding transgenic potato plants expressing dsACT in chloroplasts, in which long dsRNA are protected from processing into siRNA to easily enter the insect body through midgut receptors.54 Moreover, data from transgenic plants expressing dsRNAs targeted to insect genes that showed negative effects on insect growth and development were obtained in the laboratory or greenhouse.

for pest control. RNAi-mediated specific dsRNA for targeting specific genes for pest control has been verified in many insect species through the injection 37−39 and feeding of dsRNAs.5,13,16,40−45 For RNAi-mediated dsRNA to control pest damage in the field, transgenic plants containing dsRNA specifically targeting an insect gene are an ideal strategy compared to other approaches, including the injection and feeding of dsRNA, which can be unstable in vitro due to diverse environments containing RNase III enzymes. In the present study, we developed transgenic potato plants containing dsJHAMT to target the mRNA of LdJHAMT gene. When CPB larvae feed on the transgenic foliage, the transcriptional level of the LdJHAMT gene was knocked down, which reduced LdJHAMT protein accumulation and JH titer in the larvae. The resulting L. decemlineata larvae showed typical traits of JH deficiency, similar to other reports.22,24,26,46 Our data confirm that LdJHAMT functions as the rate-limiting enzyme in JH biosynthesis, consistent with previous reports.22−24,26,46−48 Similar results associated with JH biosynthesis were reported for other insects, such as Bombyx mori,47 Tribolium castaneum,46 D. melanogaster,24 Samia cynthia ricini,48 A. aegypti,23,25 Schistocerca gregaria,22 and Apis mellifera.49 Importantly, our results showed that plant-derived dsJHAMT ingestion reduces the LdJHAMT mRNA level and protein accumulation, resulting in typical phenotypes of JH deficiency. As shown in Figure 3, when the second- and fourthinstar larvae were fed dsJHAMT transgenic foliage, lower larval lethality and pupate rates of the surviving second- and fourthlarvae and the emerged pupae were observed compared to the control. Additionally, the tested larvae had decreased weight and a shortened developmental period. Moreover, the emerged adult females from surviving larvae laid fewer or no eggs, resulting in decreased reproduction potential, possibly due to abnormal insect growth and development. As shown in Figure 4, the female adults only laid fewer eggs compared to the control. To further evaluate the effect of plant-derived dsJHAMT ingestion on the disruption of JH biosynthesis, we used a JH analog pyriproxifen to compensate for JH deficiency by coating the transgenic foliage with the compound. The results of larvae feeding assay showed that pyriproxifen rescues insects from the negative effects of plant-derived dsJHAMT. G

DOI: 10.1021/acs.jafc.8b03914 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 6. Work model on dsRNAs high-efficiently protect transgenic potato from Leptinotarsa decemlineata in field at a large scale. The model of this work shows the dsJHAMT transgenic plant protected from pest in field at a large scale without spraying pesticide over years. X indicates the population size of insect.

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However, in the open field, efficient protection of transgenic dsRNA plants against insect pests has not been achieved. Our results showed that the transgenic dsJHAMT plants could confer high resistance to CPB in the field. Collectively, all the data suggest that female adults on the three transgenic potato lines laid fewer eggs, leading to significant reduction of reproduction potential in the field trials. Therefore, when certain methods to work were designed to control the CPB number in the early stage of potato growth, these transgenic plants containing dsJHAMT can be fully protected from CPB damage. Here, a work model of protecting the transgenic dsJHAMT plants from insect pest damage is tried to construct as a designed applicant instance (Figure 6). When the transgenic plants producing dsJHAMT are planted at large scale in the field with CPB infestation, the chemical pesticides are normally used for plant protection at the early stage of potato growth, which can remarkably reduce number of overwintering CPBs, about the rest of the 5% of insect population size according to Figures 4c and 5b, mainly due to low reproduction potential of surviving insects. The next year, the transgenic plants in the same field can be slightly damaged because of a lower population size of CPBs than last year. If the transgenic dsJHAMT plants are continuously grown in the same field, they are possibly fully protected from the CPB damage for potato production over the years. Thus, the dsRNA of LdJHAMT can high-efficiently or even completely protect transgenic potato plants from insect damage through this work model.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax/Tel: (+86) 10 64807375. *E-mail: [email protected]. Fax/Tel: (+86) 10 82109570. ORCID

Jiahe Wu: 0000-0003-0012-2174 Author Contributions #

Co-first authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Guoqing Li in Nanjing Agricultural University for his insightful opinions. This work was supported by grants from the Project for Support of Xinjiang through Science and Technology (CAS-XJ-B02), the Special Fund of Agro-Scientific Research in the Public Interest (201103026-2), and National Natural Science Foundation of China (41807404).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b03914. Primer sets used for isolation of target genes, PCR, and qPCR; screenshot on blast alignment of nucleotide identical analysis of JHAMT genes from insects deposited in NCBI data; structure of pHANNIBALdsJHAMT; damage syndromes of the transgenic potato foliages at 3 days after inoculation with second-instar CPB larvae; pyriproxifen application rescuing the negative effects of dsJHAMT transgenic foliage ingestion on larvae; exogenous pyriproxifen application compensating the dsJHAMT RNAi effects on larvae feeding on the transgenic foliages; average number of surviving CPBs and eggs 50 days after inoculation in the transgenic lines and control in 2017 (PDF) H

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